Metabolites of ecteinascidin 743 formed by human cytochrome CYP3A4

ABSTRACT

The purification and structure elucidation of several products of the metabolism of Et 743 by human cytochrome CYP3A4 have been accomplished. These compounds are abbreviated herein as “ETM” followed by a numeric value which represents the approximate molecular weight. Three compounds have been identified to date, namely ETM 305, ETM 775 and ETM 204. The structures of these ecteinascidin metabolites are as follows:

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority benefit from copending U.S.Provisional Application Serial No. 60/085,024, filed May 11, 1998, thedisclosure of which is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The ecteinascidins (herein abbreviated Et or Et's) areexceedingly potent antitumor agents isolated from the marine tunicateEcteinascidia turbinata. In particular, Et's 729, 743 and 722 havedemonstrated promising efficacy in vivo, including activity against P388murine leukemia, B16 melanoma, Lewis lung carcinoma, and several humantumor xenograft models in mice.

[0003] The isolation and characterization of natural Et 743 is taught inU.S. Pat. No. 5,089,273 which is hereby incorporated herein byreference. The preparation of synthetic Et 743 is taught in U.S. Pat.No. 5,721,362, which is hereby incorporated herein by reference.

[0004] The antitumor activities of ecteinascidin compounds, particularlyEt 729 and Et 743 are well documented in the scientific literature. Seefor example, Goldwasser et al., Proceedings of the American Associationfor Cancer Research, 39: 598 (1998); Kuffel et al., Proceedings of theAmerican Association for Cancer Research, 38: 596 (1997); Moore et al.,Proceedings of the American Association for Cancer Research, 38: 314(1997); Mirsalis et al., Proceedings of the American Association forCancer Research, 38: 309 (1997); Reid et al., Cancer Chemotherapy andPharmacology, 38: 329-334 (1996); Faircloth et al., European Journal ofCancer, 32A, Supp. 1, pp. S5 (1996); Garcia-Rocha et al., BritishJournal of Cancer, 73: 875-883 (1996); Eckhardt et al., Proceedings ofthe American Association for Cancer Research, 37: 409 (1996); Hendrikset al., Proceedings of the American Association for Cancer Research, 37:389 (1996); the disclosures of which are hereby incorporated herein byreference.

[0005] Ecteinascidin 743 (Et 743) has the following structure:

[0006] In view of the impressive antitumor activities of this class ofcompounds, the search continues for related structures that may possessequal or higher levels of antitumor activity. The present invention,which is directed to the isolation and characterization of naturalmetabolites of Et 743, is a result of these continued studies.

SUMMARY OF THE INVENTION

[0007] The purification and structure elucidation of several products ofthe metabolism of Et 743 by human cytochrome CYP3A4 have beenaccomplished. These compounds are abbreviated herein as “ETM” followedby a numeric value which represents the approximate molecular weight.

[0008] For example, ETM 305 and ETM 775 were isolated from a metabolicmixture obtained from a biochemical study performed by the AnalyticalChemistry Department at PharmaMar, Spain. A similar metabolic studycarried out by the Mayo Clinic led to the identification of ETM 204. Thestructures of these ecteinascidin metabolites are as follows:

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The present invention may be better understood by reference tothe drawings accompanying this specification, wherein:

[0010]FIG. 1 is the ¹H NMR spectrum (500 MHz) of ETM-SiOH-1 (non-polarimpurity) in CDCl₃;

[0011]FIG. 2 is the HPLC chromatogram of ETM-SiOH-4 (ETM 775);

[0012]FIG. 3 is the HPLC chromatogram of ETM-SiOH-3 (ETM 305);

[0013]FIG. 4 is the HPLC chromatogram of ETM-SiOH-2 (trace metabolites);

[0014]FIG. 5 is the LRFAB mass spectrum of ETM 305 in M.B. (magicbullet⁴);

[0015]FIG. 6 is the ESI mass spectrum of ETM 305;

[0016]FIG. 7 is the ¹H NMR spectrum (750 MHz) of ETM 305 in CD₃OD;

[0017]FIG. 8 is the FAB/MS/MS spectrum of ETM 305;

[0018]FIG. 9 is the UV spectrum of ETM 305;

[0019]FIG. 10 is the UV spectrum of ETM;

[0020]FIG. 11 is the LRFAB mass spectrum of ETM 775 in M.B.;

[0021]FIG. 12 is the ESI mass spectrum of ETM 775 (positive mode);

[0022]FIG. 13 is the ESI mass spectrum of ETM 775 (negative mode);

[0023]FIG. 14 is the FAB/MS/MS spectrum of ETM 775 (m/z 138-302);

[0024]FIG. 15 is the FAB/MS/MS spectrum of ETM 775 (m/z 440-620);

[0025]FIG. 16 is the ¹H NMR spectrum (750 MHz) of ETM 775 in CD₃OD;

[0026]FIG. 17 is the UV spectrum of ETM 775;

[0027]FIG. 18 is the HPLC choromatogram of ETM 305;

[0028]FIG. 19 is the UV spectrum of ETM 305;

[0029]FIG. 20 is the ESI mass spectrum of ETM 305;

[0030]FIG. 21 is the ESI mass spectrum of ETM 204;

[0031]FIG. 22 is the ¹H NMR spectrum (500 MHz) of ETM 204 in CD₃OD; and

[0032]FIG. 23 is the ESI/MS/MS spectrum of ETM 204.

DETAILED DESCRIPTION OF THE INVENTION

[0033] I. Et 743 Metabolic Study

[0034] A. Preparation of Metabolic Mixture—ETM:

[0035] Et-743 (50 μM) was incubated with 0.4 mg/ml of humanlymphoblast-expressed CYP3A4 isoform (Gentest Corporation, Woburn,Mass.) in 0.1 M Tris-HCl buffer (pH 7.4) containing an NADPH generatingsystem (0.4 mM NADP⁺, 25 mM glucose-6-phosphate, 0.5 U/mlglucose-6-phosphate dehydrogenase and 3.3 mM magnesium chloride). Afterfour (4) hours at 37° C., the reaction was stopped with ice coldacetonitrile and the solids removed by centrifugation (12,000 g, 4min.). Supernatants were analyzed by HPLC.

[0036] B. Purification of ETM 305 and ETM 775

[0037] 2.6 mg of ETM (generated as in A, above) was dissolved in a smallamount of CHCl₃ and loaded into a silica gel column (8×100 mm glasscolumn filled with a silica gel/CHCl₃ slurry). First, the column waseluted with CHCl₃ followed by CHCl₃/MeOH mixtures (98, 96, 94, 92 and90%). A total of ten test tubes were collected (3 mL each) and combinedon the basis of TLC to yield four fractions (Table 1). The less polarand non-cytotoxic fraction (ETM-SiOH-1, 2 mg) consisted of a lipid notstructurally related to Et 743 as revealed by the ¹H NMR spectrum (FIG.1).

[0038] The remaining cytotoxic fractions were further purified by HPLC(Phenomenex-Ultracarb ODS, 10 μm, 10×150 mm, 3:1 MeOH/H₂O 0.02 M NaCl, 1mL/min., Da Detection: 210, 220, 254 and 280 nm). The most polarfraction (ETM-SiOH-4, 0.2 mg) yield 0. 1 mg of ETM 775 (FIG. 2).ETM-SiOH-3 yield 0.3 mg of ETM 305 (FIG. 3), and ETM-SiOH-2 consisted ofa complex mixture of trace metabolites (FIG. 4). TABLE 1 ETM-SiOHfractions: R_(f), weight and cytoxic activity. L1210 growth inhibition(%) ID# Test tube # R_(f) ^(a) Weight at 500 ng/mL ETM-SiOH 1 1 0.9 2.0mg  0 ETM-SiOH 2 2 0.5, 0.7 0.3 mg  80^(b) ETM-SiOH 3 4-5 0.5 0.4 mg 30ETM-SiOH 4 6 0.3 0.2 mg  3

[0039] C. The Structure of ETM 305.

[0040] ETM 305 (IC₅₀ 0.2 μm/mL vs L12 10 cells) showed a molecular ionat 306.0977 by HRFAB/MS (FIG. 5). This data is in agreement with themolecular formula C₁₅H₁₆NO₆ (Δ0.1 mmu). ESI/MS analysis confirmed themolecular weight of ETM 305 (FIG. 6); a molecular ion at m/z 306 wasobserved together with its sodium adduct (m/z 328). The ¹H NMR spectrumof ETM 305 (FIG. 7) was very important for the structural assignment.Resonances at δ2.04, 2.28 and 6.09 were almost identical to those ofMe-6 (δ2.03), —OCOCH₃ (δ2.29) and the dioxy-methylene protons (δ6.11 and6.01) in Et 743,¹ respectively.

[0041] In addition, it was observed resonances corresponding to a—CH═CH—NHCHO unit (δ7.09, d, 1H, J=15 Hz; δ6.19, d, 1H, J=15 Hz; δ8.04,s, 1H),² and an additional methyl group (δ2.52, s, 3H). The chemicalshift of this methyl group match pretty well wit that of the methylgroup on acetophenone ³(δ2.55). It is interesting to note that the ¹HNMR spectrum of ETM 305 consisted of two sets of resonances (4:1 ratio)due to rotational conformers around the —NH—CHO bond The ¹H NMR datatogether with the MS data suggested that ETM 305 had the B-unit aromaticring system of Et 743 attached to a vinyl-formamide unit and to a methylketone as shown in Scheme 1. FAB/MS/MS on m/z 306 supported the proposedstructure (FIG. 8).

[0042] D. The Structure of ETM 775.

[0043] ETM 775 (IC₅₀ 0.2 μg/mL vs L1210 cells) showed a molecular ion at776.2489 by HRFAB/MS (FIG. 11). This data is in agreement with themolecular formula C₃₉H₄₂N₃O₁₂S (Δ0.0 mmu) which indicated that ETM 775is an oxidation product of Et 743. Both, positive and negative modeESI/MS spectra confirmed the molecular weight of ETM 775 (FIGS. 12 and13). Because of the limited amount of ETM 775, the structural assignmentwas carried out mainly by interpretation of its mass spectral data.FABMS/MS on M+H of ETM 775 (m/z 776) was critical in assigning thelocation of the extra oxygen was located on N-2 in the form of anN-oxide as revealed by peaks at m/z 276 and 260 (276 -oxygen). Afragment ion at m/z 232, not observed in Et 743, suggested that thecarbinol amine oxygen was oxidized to the amide (Scheme 3). Thestructures of the A- and C-units in ETM 775 remained intact as revealedby the presence of the characteristic mass spectral peaks at m/z 204(A-unit), and m/z 224 and 250 (C-unit).¹ Both, the 750 750 Mhz ¹H NMR(FIG. 16) and the UV (FIG. 17) spectra resembled those of Et 743.¹

[0044] II. Et 743—Mayo Metabolic Study

[0045] A. M1 Metabolite (ETM 305).

[0046] The ETM sample was filtered through a C18 sep-pack and the eluant(3:1 MeOH/H₂O) concentrated under a nitrogen stream. Purification of theresulting residue by HPLC (same conditions as described above) revealedthe presence of a compound with a retention time identical to that ofETM 305 (FIG. 18). Both, the UV (FIG. 19) and ESI/MS (FIG. 20) spectraof M1 were identical to that of ETM 305. Thus, it was concluded that theM1 metabolite had the same chemical structure as ETM 305.

[0047] B. M2 Metabolite (ETM 204).

[0048] The provided sample was filtered through a C18 sep-pack and theeluant (3:1 MeOH/H₂O) concentrated under a nitrogen stream and theresulting residue analyzed by FAB/MS, ESI/MS and ¹H NMR.

[0049] C. The Structure of ETM 204 (M2).

[0050] ETM 204 showed a molecular ion at 204.1024 by HRFAB/MS. This datais in agreement with the molecular formula C₁₂H₁₄NO₂ (Δ0.0 mmu). ESI/MSanalysis confirmed the molecular weight as 204 (FIG. 21). The molecularformula matched with the molecular formula of the a-unit in Et 743.Thus, the chemical structure of ETM 204 was proposed to be the aromaticammonium salt derivative shown in Scheme 3. This simple compound (aswell as the other metabolites) can easily be monitored to assay thebreakdown of Et 743 in vivo.

[0051] A ¹H NMR spectrum (FIG. 22) of ETM 204 showed resonances thatsupported the proposed structure: four aromatics signals (δ9.2, s; δ7.8,d, J=5 Hz, and δ6.8, s) and three methyl singlets (δ4.2, δ3.9 and δ2.4)The ESI/MS/MS of ETM 204 (FIG. 23) showed a prominent peak ion at 189corresponding to the apparent loss of the N-methyl group (204-CH₃).

[0052] Biological Studies of ETM-305 and ETM-775:

[0053] Compounds ETM-305 and ETM-775 have been assayed employingstandard protocols for the following tumor cell lines; P-388 (murineleukemia); A-549 (human lung carcinoma); HT-29 (human colonadenocarcinoma); and MEL-28 (human malignant melanoma). See, forexample, Bergeron et al., Biochem. Biophys. Res. Comm., 1984, 121 (3)848-854 and Schroeder et al., J. Med. Chem., 1981, 24 1078-1083. Theseresults are shown below in Table 2: TABLE 2 Cell Line & Activity IC₅₀(μg/ml) Compound: P-388 A-549 HT-29 MEL-28 ETM-305 0.5  0.5  0.5  0.25ETM-775 0.01 0.01 0.01 0.01

[0054] Methods of Treatment

[0055] The present invention includes bioactive compounds, andaccordingly, an embodiment of the present invention is directed tomethods of treatment using such compounds. As described above, thecompounds of the present invention have exhibited in vitro cytoxicityagainst tumor cell lines. It is anticipated that these in vitroactivities will likewise extend to in vivo utility.

[0056] These compounds have been isolated in substantially pure form,i.e., at a purity level sufficient to allow physical and biologicalcharacterization thereof. These compounds have been found to possessspecific antitumor activities and as such they will be useful asmedicinal agents in mammals, particularly in humans. thus, anotheraspect of the present invention concerns pharmaceutical compositionscontaining the active compounds identified herein and methods oftreatment employment such pharmaceutical compositions.

[0057] As described above, the active compounds of the present inventionexhibit antitumor activity. thus, the present invention also provides amethod of treating any mammal affected by a malignant tumor sensitive tothese compounds, which comprises administering to the affectedindividual a therapeutically effective amount of an active compound ormixture of compounds, or pharmaceutical compositions thereof. Thepresent invention also relates to pharmaceutical preparations, whichcontain as active ingredient one or more of the compounds of thisinvention, as well as the processes for its preparation.

[0058] Example of pharmaceutical compositions include any solid(tablets, pills, capsules, granules, etc.) or liquid (solutions,suspensions of emulsions) with suitable composition or oral, topical orparenteral administration, and they may contained the pure compound orin combination with any carrier of other pharmacologically activecompounds. These compositions may need to be sterile when administeredparenterally.

[0059] The terms “unit dose” as it pertains to the present inventionrefers to physically discrete units suitable as unitary dosages foranimals, each unit containing a predetermined quantity of activematerial calculated to produce the desired antitumor effect inassociation with the required diluent; i.e., carrier, or vehicle. Thespecifications for the novel unit dose of this invention are dictated byand are directly dependent on (a) the unique characteristics of theactive material and the particular antitumor effect to be achieved, and(b) the limitations inherent in the art of compounding such activematerial for antitumor use in animals.

[0060] Unit dosage forms are typically prepared from the frozen or driedactive compound (or salts thereof by dispersement in a physiologicallytolerable (i.e., acceptable) diluent or vehicle such as water, saline orphosphate-buffered saline to form an aqueous composition. Such diluentsare well known in the art and are discussed, for example, in Remington'sPharmaceutical Sciences, 16th Ed., Mack Publishing Company, Easton, Pa.(1980) at pages 1465-1467.

[0061] Dosage forms can also include an adjuvant as part of the diluent.Adjuvants such as complete Freund's adjuvant (CFA), incomplete Freund'sadjuvant (IFA) and alum are materials well known in the art, and areavailable commercially from several sources.

[0062] The quantity of active compound to be administered depends, interalia, on the animal species to be treated, the subject animal's size,the size of the tumor (if known), the type of tumor (e.g., solid)present, and the capacity of the subject to utilize the active compound.Precise amounts of active compound required to be administered depend onthe judgment of the practitioner and are peculiar to each individual,particularly where humans are the treated animals. Dosage ranges,however, can be characterized by a therapeutically effective bloodconcentration and can range from a concentration of from about 0.01 μMto about 100 μM, preferably about 0.1 μM to 10 μM.

[0063] Suitable regimes for initial administration and boosterinjections are also variable, but are typified by an initialadministration followed by repeated doses at one or more hour intervalsby a subsequent injection or other administration. Alternatively,continuous intravenous infusion sufficient to maintain a therapeuticallyeffective concentration in the blood are contemplated.

REFERENCES

[0064] The following background references are provided to assist thereader in understanding this invention. To the extent necessary, thecontents are hereby incorporated herein by reference.

[0065] 1. A) Rinehart et al., J. Org. Chem. 1990, 55, 4512. B) Rinehartet al., J. Am. Chem. Soc., 1996, 118 9017.

[0066] 2. Herbert et al., J. Chem. Soc. Perkin Trans. I, 1987, 1593.

[0067] 3. Pretsch et al. Tables of Spectral Data for StructureDetermination of Organic Compounds; Springer-Verla: Berlin, 1989; p.H125.

[0068] 4. Rinehart et al., Biochem. Res. Commun., 1984, 124, 350.

[0069] The present invention has been described in detail, including thepreferred embodiments thereof. However, it will be appreciated thatthose skilled in the art, upon consideration of the present disclosure,may make modifications and/or improvements on this invention and stillbe within the scope and spirit of this invention.

What is claimed is:
 1. Substantially pure ETM-305, having the followingstructure:


2. Substantially pure ETM-204, having the following structure:


3. Substantially pure ETM-775 having the following structure:


4. A method of treating mammalian leukemia in patients in need of suchtreatment, said method comprising administering an effective amount ofETM-305 to said patient in unit dosage form.
 5. A method of treatingmammalian leukemia in patients in need of such treatment, said methodcomprising administering an effective amount of ETM-775 to said patientin unit dosage form.
 6. A method of treating mammalian lung carcinoma inpatients in need of such treatment, said method comprising administeringan effective amount of ETM-305 to said patient in unit dosage form.
 7. Amethod of treating mammalian lung carcinoma in patients in need of suchtreatment, said method comprising administering an effective amount ofETM-775 to said patient in unit dosage form.
 8. A method of treatingmammalian colon adenocarcinoma in patients in need of such treatment,said method comprising administering an effective amount of ETM-305 tosaid patient in unit dosage form.
 9. A method of treating mammaliancolon adenocarcinoma in patients in need of such treatment, said methodcomprising administering an effective amount of ETM-775 to said patientin unit dosage form.
 10. A method of treating mammalian malignantmelanoma in patients in need of such treatment, said method comprisingadministering an effective amount of ETM-305 to said patient in unitdosage form.
 11. A method of treating mammalian malignant melanoma inpatients in need of such treatment, said method comprising administeringan effective amount of ETM-775 to said patient in unit dosage form. 12.A pharmaceutical composition comprising ETM-305 and a pharmaceuticallyacceptable carrier, diluent or excipient.
 13. A pharmaceuticalcomposition comprising ETM-775 and a pharmaceutically acceptablecarrier, diluent or excipient.
 14. A method of assaying the humancytochrome CYP3A4 metabolism of ecteinascidin 743, comprising monitoringa test sample for the presence of one or more metabolites selected fromthe group consisting of ETM-204, ETM-305 and ETM-775.